The invention is directed to surface enhanced spectroscopy-active composite nanoparticles, methods of manufacture of the particles, and uses of the particles (including their use as molecular or cellular optical tags). More particularly, it is directed to the area of submicron-sized tags or labels that can be covalently or non-covalently affixed to entities of interest for the purpose of quantitation, location, identification, and/or tracking.
When light is directed onto a molecule, the vast majority of the incident photons are elastically scattered without a change in frequency. This is termed Rayleigh scattering. However, the energy of some of the incident photons (approximately 1 in every 107 incident photons) is coupled into distinct vibrational modes of the molecule""s bonds. Such coupling causes some of the incident light to be inelastically scattered by the molecule with a range of frequencies that differ from the range of the incident light. This is termed the Raman effect. By plotting the frequency of such inelastically scattered light against its intensity, the unique Raman spectrum of the molecule under observation is obtained. Analysis of the Raman spectrum of an unknown sample can yield information about the sample""s molecular composition.
The incident illumination for Raman spectroscopy, usually provided by a laser, can be concentrated to a small spot if the spectroscope is built with the configuration of a microscope. Since the Raman signal scales linearly with laser power, light intensity at the sample can be very high in order to optimize sensitivity of the instrument. Moreover, because the Raman response of a molecule occurs essentially instantaneously (without any long-lived highly energetic intermediate states), photobleaching of the Raman-active moleculexe2x80x94even by this high intensity lightxe2x80x94is impossible. This places Raman spectroscopy in stark contrast to fluorescence spectroscopy, where photobleaching dramatically limits many applications.
The Raman effect can be significantly enhanced by bringing the Raman-active molecule(s) close ( less than 50 xc3x85) to a structured metal surface; this field decays exponentially away from the surface. Bringing molecules in close proximity to metal surfaces is typically achieved through adsorption of the Raman-active molecule onto suitably roughened gold, silver or copper or other free electron metals. Surface-enhancement of the Raman activity is observed with metal colloidal particles, metal films on dielectric substrates, and with metal particle arrays. The mechanism by which this surface-enhanced Raman scattering (SERS) occurs is understood, and is thought to result from a combination of (i) surface plasmon resonances in the metal that enhance the local intensity of the light, and; (ii) formation and subsequent transitions of charge-transfer complexes between the metal surface and the Raman-active molecule.
SERS allows detection of molecules attached to the surface of a single gold or silver nanoparticle. A Raman enhancing metal that has associated or bound to it a Raman-active molecule(s) is referred to as a SERS-active nanoparticle. Such SERS-active nanoparticles can have utility as optical tags. For example, SERS-active nanoparticles can be used in immunoassays when conjugated to an antibody against a target molecule of interest. If the target of interest is immobilized on a solid support, then the interaction between a single target molecule and a single nanoparticle-bound antibody could be detected by searching for the Raman-active molecule""s unique Raman spectrum. Furthermore, because a single Raman spectrum (from 100 to 3500 cmxe2x88x921) can detect many different Raman-active molecules, SERS-active nanoparticles may be used in multiplexed assay formats.
SERS-active nanoparticles offer the potential for unprecedented sensitivity, stability, and multiplexing functionality, when used as optical tags in chemical assays. However, SERS-active nanoparticles made from metals present formidable practical problems when used in such assays. Metal nanoparticles are exceedingly sensitive to aggregation in aqueous solution; once aggregated, it is not possible to re-disperse them. In addition, the chemical compositions of some Raman-active molecules are incompatible with the chemistries used to attach other molecules (such as proteins) to metal nanoparticles. This restricts the choices of Raman-active molecules, attachment chemistries, and other molecules to be attached to the metal nanoparticle.
The most significant problem with the use of metal nanoparticles as Raman tags is the similarity of the Raman spectra of molecules to be coupled to the nanoparticles. For example, in a multiplexed sandwich immunoassay, the Raman spectra of the secondary antibodies to which the nanoparticles are attached would be highly similar, and thus impossible to deconvolute. Moreover, the parts of the secondary antibodies that are different, i.e., the antigen-binding domains, are typically too far away from the metal surface to be significantly enhanced.
The prior art teaches that molecules themselves can be used as Raman tags, provided that their Raman scattering cross section is sufficiently large. Thus, direct attachment of dyes, for example, to antibodies, allows them to be used as tags for immunoassays. This approach, however, suffers from extremely significant limitations: the molecular structures/features that give rise to intense Raman spectra (e.g. polarizability, aromaticity, conjugation, heteroatoms, and most significantly, significant absorption cross section) also give rise to complex Raman spectra. The use of molecular Raman tags requires very high extinctions in the visible region of the spectrum to access resonance Raman scattering, which increases the Raman signal by up to three orders of magnitude. There is a fundamental physical incompatibility between molecules that absorb visible light well and those that exhibit simple Raman spectra. Thus, the Raman spectra of the dyes described above are exceedingly complex, and it has not been possible to multiplex these assays.
A second fundamental problem with Raman-based tags is the weakness of the Raman signal; it is not possible to detect single molecules (or even thousands of molecules) by Raman without using surface enhancement. Ideally, one would like a tag that exhibits the enhancement factors associated with SERS and the ability to attach such a tag to a freely diffusing species (which would clearly not be possible with macroscopic SERS-active surfaces).
It is an object of this invention to provide a solution to the abovementioned problems encountered when using Raman scattering entities as optically-addressable labels or tags, especially in chemical or biomolecular assays. It is a further object of the invention to provide a panel of at least 20 different SERS-active nanoparticles for use as xe2x80x9ccleavelessxe2x80x9d optical tags in bead-based combinatorial chemical syntheses. It is a further object of this invention to describe an optical detection system for multiplexed assays.
The present invention is directed to surface enhanced spectroscopy-active composite nanoparticles, including SERS-active composite nanoparticles (SACNs). Also included within the scope of this invention are methods of manufacture of the particles, and uses of the particles (including their use as molecular or cellular optical tags). The submicron-sized-tags or labels of the invention can be covalently or non-covalently affixed to entities of interest(that may range in size from molecules to macroscopic objects) for the purpose of quantitation, location, identification, and/or tracking.
The present invention overcomes the problems encountered when using a spectroscopy-active species, such as a Raman scattering species, as an optical tag. The invention provides novel SES-active composite nanoparticles, including SERS-active composite nanoparticles (SACNs). Such nanoparticles each comprise a SES-active metal nanoparticle, a submonolayer, monolayer, or multilayer of spectroscopy-active species in close proximity to the metal surface, and an encapsulating shell comprising a polymer, glass, or any other dielectric material. This places the spectroscopy-active molecule (alternately referred to herein as the xe2x80x9canalytexe2x80x9d; not to be confused with the species in solution that is ultimately being quantified) at the interface between the metal nanoparticle and the encapsulant.
In preferred embodiments, the encapsulant is glass. The resulting glass-coated analyte-loaded nanoparticles (GANs) retain the activity of the SES-active metal nanoparticles, but tightly sequester this activity from the exterior surface of the nanoparticle. Thus, in the case of surface active Raman scattering (SERS), the resulting GANs exhibits SERS activity, but the Raman-active analyte is located at the interface between the metal nanoparticle and the encapsulant.
The analyte molecule can be chosen to exhibit extremely simple Raman spectra, because there is no need for the species to absorb visible light. This, in turn, allows multiple GANs particles, each with different analyte molecules, to be fabricated such that the Raman spectrum of each analyte can be distinguished in a mixture of different types of GANs particles.
GANs are easily handled and stored. They are also aggregation resistant, stabilized against decomposition of the analyte in solvent and air, chemically inert, and can be centrifuged and redispersed without loss of SERS activity.
Most importantly, the glass shells of GANs may be readily derivatized by standard techniques. This allows GANs to be conjugated to molecules (including biomolecules such as proteins and nucleic acids) or to solid supports without interfering with the Raman activity of the GANs. Unlike metal nanoparticles, GANs can be evaporated to dryness, and then completely redispersed in solvent. Using the techniques provided herein, it is possible to fabricate GANs that are individually detectable using SERS.
The SACNs provided by the present invention are uniquely identifiable nanoparticles. They can be used in virtually any situation in which it is necessary to label molecules or objects (including beads and other types of solid support), with an optical tag. Biomolecules can be conjugated readily to the exterior of SACNs by standard techniques, thereby allowing the particles to function as optical tags in biological assays. SACNs can be used in virtually any assay that uses an optical tag, such as a fluorescent label. However, as optical tags, SACNs have several distinct advantages over fluorescent labels. These advantages include vastly more sensitive detection, chemical uniformity, and the absolute resistance of the SERS activity to photobleaching or photodegradation. A further benefit of using SACNs as optical tags is the ease with which individual SACNs having different SERS-activities may be resolved from one another. At least twenty different SACNs are resolvable from one another using a simple Raman spectroscope. This enables multiplexed assays to be performed using a panel of different SACNs, each having a unique and distinguishable SERS-activity.
In addition, SACNs can serve as novel xe2x80x9ccleavelessxe2x80x9d optical tags in bead-based combinatorial chemical syntheses. In this embodiment, each synthetic step in the scheme can be accompanied by the conjugation of a unique SACN to the bead. The reaction history of the bead, and hence the identity of the synthesized compound, can then be determined by reading the SERS spectrum of the bead, without first requiring that the SACNs are cleaved from the bead.